Bimolecular Complementation to Visualize Filovirus VP40-Host Complexes in Live Mammalian Cells: Toward the Identification of Budding Inhibitors
Figure 2
BiMC detecting eVP40-Tsg101 interactions and trafficking of Tsg101/eVP40 complex. (a) Human 293T cells grown on coverslips in 6-well plates were transfected with NYFP-Tsg101 + CYFP-eVP40-WT (a) or NYFP-Tsg101 + CYFP-eVP40-ΔPT/PY. (b) Cells were washed, fixed, and examined for YFP fluorescence by confocal microscopy at 24 hours p.t. Cells were mock-transfected, or transfected with 20 nM of Tsg101-specific or random siRNA. Twenty four hours later, cells were mock-transfected or transfected with the same amount of siRNA along with NYFP-Tsg101 + CYFP-eVP40. After an additional 24 hours, cells were examined for YFP fluorescence as described above. (b) Colocalization eVP40-Tsg101 complexes with pericentrin. BiMC assay showing colocalization between pericentrin-B and YFP fluorescence from 293T cells expressing NYFP-Tsg101 + CYFP-eVP40-WT at 6 hours p.t. (top panel) and 24 hours p.t. (bottom panel). (c) DIC images of a single 293T cell co-expressing NYFP-Tsg101 + CYFP-eVP40-WT at 4, 8, 12, 16, and 20 hours p.t. Blue arrows indicate Tsg101/eVP40 complex accumulation at the plasma membrane.
